Each year, 100,000 Americans perish due to untreatable bacterial infections. The societal benefits of new antibiotic compounds that are effective against numerous multiple drug resistant pathogens would be significant. The best possible source for new antibiotic scaffolds with potentially novel mechanisms of action is within natural environments, particularly soils, which have the greatest diversity of microbial life. Our recent research advances the science of metagenomics, the cloning of DNA from entire microbial communities, to discover novel antibiotics and identify the best lead candidates for clinical development. A team of scientists from academia and Lucigen Corporation have united four key technological breakthroughs that together resulted in the next generation metagenomic library. This library combined 1) an improved methodology for the isolation and purification of high molecular weight genomic DNA from soil microorganisms; 2) a new broad host range shuttle vector for enhanced expression of cloned DNAs; 3) a random shear cloning method to produce very large insert sizes (>100 kb); and 4) a rapid and improved screening method to identify antibiotic-producing clones within a metagenomic library. The library was screened against a clinical isolate of methicillin-resistant Staphylococcus aureus (MRSA), resulting in the identification of 28 metagenomic clones that produce anti-MRSA compounds, a hit rate of 1 in 685. Twelve of these anti-MRSA clones were analyzed by sequencing and found to have very large insert sizes (average 113.5 kb) and novel genetic diversity not encountered before. Moreover, one of the clones was found to produce a novel metabolite. These results are 10-100 fold more efficient than previous efforts. However, the primary bottleneck now is elucidating the structure of these compounds. Conventional technologies often require many man-years of effort to elucidate a single structure. This Phase I research proposal endeavors to improve that level by approximately ten-fold. In fact we propose to find new small molecules from this next generation metagenomics library by screening for structures. If successful the rate of natural product discovery could be accelerated many fold compared to functional based screens. PUBLIC HEALTH RELEVANCE: In the fight against microbial infectious disease we are losing ground due to the development of antibiotic resistance and our inability to find replacement drugs. The loss of life and the burden of treatment is a significant public health threat to American citizens. The proposed research unleashes a new set of tools for drug discovery that is 10-100 times more efficient than conventional technologies.